Growth inhibitions of selective mycoplasmas

ABSTRACT

The use of certain heparinoid compounds to selectively inhibit the growth of mycoplasmas is described. The invention provides a practical means for facilitating the selective identification of mycoplasmas. The identification of mycoplasmas isolated from man is especially useful as a diagnostic aid in prescribing treatment for diseases caused by these microorganisms. Additionally, the invention provides a means of preventing the growth of mycoplasmas in culture media especially media used for tissue cultures in which mycoplasmas frequently occur as undesirable contaminants.

United States Patent Cekoric, Jr. et. al.

[ 1 June 6, 1972 [54] GROWTH INHIBITIONS OF SELECTIVE MYCOPLASMAS [72] inventors: Thomas Cekoric, Jr.; George Evans, both of Hopatcong; Ronald Searcy, Upper Montclair, all of NJ.

[73] Assignee: Hoffman-LaRoche, lnc., Nutley, NJ.

[22] Filed: Oct. 4, 1968 [211 App]. No.: 765,028 I Related U.S. Application Data [63] Continuation-impart of Ser. No. 676,976, Oct. 20,

1967, abandoned.

521 u.s.c1. ....19s/103.s,19s/100 s1 1m.c1. ..Cl2k 1/06 58] FieldofSearch ..195/100,103.5;424/180 [56] References Cited UNlTED STATES PATENTS 2,694,058 I 1/1954 Berger ..260/209.6

OTHER PUBLICATIONS Stuart; J. 0111. Path." Vol. 1. 1 1943 pp. 311- 314 Primary Examiner-A. Louis Monacell Assistant Examiner-Max D. Hensley Attorney-Samuel L. Welt, Jon S. Saxe, Bernard S. Leon and Gerald S. Rosen [57] ABSTRACT The use of certain heparinoid compounds to selectively inhibit the growth of mycoplasmas is described.

Additionally, the invention provides a means of preventing the growth of mycoplasmas in culture media especially media used for tissue cultures in which mycoplasmas frequently occur as undesirable contaminants.

10 Claims, No Drawings GROWTH INHIBITIONS OF SELECTIVE MYCOPLASMAS RELATED APPLICATION BACKGROUND OF THE INVENTION Mycoplasmas are a group of microorganisms which are intermediate in size between bacteria and viruses. Many mycoplasma species have already been identified. Certain of these have been characterized as human strains. However, an even greater number have been characterized as animal strains. Included among the human mycoplasma species which, to date, have been characterized are, for example, Mycaplarma homim's, type I and Mycoplasma hominis, type 2 (M. anhritidis), Mycoplasma salivarium, Mycoplasma fermenrans, Mycoplasma orale, types 1 and 2 and Mycoplasma pneumoniae. The latter species of mycoplasma is considered by many to be the etiologic agent in primary atypical pneumonia. Furthermore, the species M. hominis, type 1 has been recovered from the genitourinary tract, and its frequent occurrence in association with venereal disease, non-bacterial urethritis, cervicitis and other inflammatory diseases of the genital tract, has been reported as well as its association with exudative pharyngitis. Mycoplasma pulmonis (Negroni agent), while not indigenous to man, has been isolated from tissue cultures inoculated with specimens from leukemia patients. Likewise, M. hominis, type 2 which is occasionally isolated from human specimens, has been shown to be identical to a rodent mycoplasma, M. anhn'tidis. The rapid identification of mycoplasmas becomes extremely important in initiating the proper treatment of conditions of which mycoplasmas are .the causative agent since they are resistant to many of the antibiotics and chemotherapeutic agents used for bacterial infec- UOIIS.

In recent years, it has been reported that mycoplasmas are present, as contaminants, in tissue cultures such as are used in the metabolic studies of cells or in the propagation of viruses. A prime contaminant has been identified as M. hominis, type 1. The occurrence of mycoplasma in tissue cultures furnishes a potential source for an erroneous interpretation of results since the interpretation invariably presumes that cultures are devoid of microbial contaminants.

Techniques and methods for isolating, identifying and inhibiting the growth of mycoplasmas, particularly the human strains, have become, therefore, important in the preparation and use of tissue cultures.

BRIEF SUMMARY OF THE INVENTION The present invention is predicated upon the finding that certain heparinoid compounds, hereinafter specifically identified, selectively inhibit the growth of certain mycoplasma species. By using these heparinoid compounds in combination, groups of human mycoplasmas can be identified. In its broadest embodiment, the invention provides a means for facilitating the identification of 'various mycoplasma species derived from a human source. In one of its more important embodiments, the invention provides a method for the specific identification of M. horru'nis, type I, M. salivan'um and M. fermentans.

The heparinoid compounds which are used in the practice of the invention are as follows: the polyanetholsulfonicacid sodium salt; the sodium salt of sulfated polymannuronic acid; the sodium salt of sulfated polymannuronic acid methyl glycoside; and the sodium salt of sulfated polyglucuronic acid methyl ester. The polyanethol sulfonic acid sodium salt and the sodium salt of sulfated polymannuronic acid methyl glycoside are known compounds and processes for their production are disclosed in the literature. Although not a part of the present invention, the production of the sodium salt of sulfated polymannuronic acid and of the sodium salt of sulfated polyglucuronic acid methyl ester will be described hereinafier for the sake of completeness of disclosure.

While the invention will be described herein with particular reference to the use of sodium salts of the various compounds, it is to be understood that the. invention is not necessarily limited to the use of such salts. Any non-toxic salt of the various compounds, which is soluble in water or saline, can be employed.

DETAILED DESCRIPTION OF THE INVENTION The ability of the aforementioned heparinoid compounds to inhibit selectively the growth of mycoplasmas has been studied by two techniques. In the first technique, paper discs, for example, discs having a diameter of from about 6 mm. to about 8 mm. are moistened with a solution of the heparinoid compound. The moistened disc is then placed, for example, on

an agar plate which contains an appropriate growth medium. The growth medium has previously been inoculated with the mycoplasma isolate to be studied. The plates are incubated in a suitable environment, e.g., at 37 C. in a moist chamber containing an atmosphere of air or a mixture of percent nitrogen and 5 percent carbon dioxide, and observed over a period of time for the appearance of microscopic colonies of the microorganism. If the heparinoid compound inhibits the growth of the mycoplasma, a zone of inhibition which can be observed under the microscope will surround the disc.

in the second technique for evaluating the inhibitory properties of the heparinoid compounds to the growth of mycoplasmas, the heparinoid compound is introduced directly into the growth medium. The container which contains the growth medium, following inoculation of the medium with the mycoplasma isolate and suitable incubation, e.g., at 37 C. in a moist chamber containing an atmosphere of air or a mixture of 95 percent nitrogen and 5 percent carbon dioxide, is observed to detect the presence or absence of growth. Growth of the culture may be detected by direct observation under the microscope, by subculture or by the production of acid from the fermentation of glucose.

The present method for identifying mycoplasmas in an isolate utilizes a combination of the foregoing techniques. It has been found that the sodium salt of polyanethol sulfonate and the sodium salt of sulfated polymannuronic acid were each active mycoplasma inhibitors when tested by the disc method. Thus, for example, the exposure of various mycoplasma species to discs saturated with the sodium salt of polyanethol sulfonate resulted in the growth inhibition of the following named mycoplasmas:

M. hominis, type 1 M. hominis, type 2 M. pulmom's M. fermentans M. omle', type 1 M. omle, type 2 The sodium salt of polyanethol sulfonate did not, however, inhibit the growth of the mycoplasma species M. salivarium.

The exposure of various mycoplasma species to discs saturated with the sodium salt of sulfated polymannuronic acid resulted in the growth inhibition of the following named mycoplasmas:

M. hominis, yp I M. pulmom's M. fermenram M. omle, type 2 The sodium salt of sulfated polymannuronic acid did not, however, inhibit the growth of M. horninis, type 2; M. salivarium; M. omle, type 1; or M. pneumoniae.

It has been found that two of the heparinoid compounds,

named heretofore, do not manifest activity as mycoplasma growth inhibitors when tested by the disc method. However, it has been found that the compounds are selective growth inhibitors when incorporated directly in the medium in which the microorganism is grown. The heparinoids which inhibit the growth of certain mycoplasmas when incorporated directly into the growth medium are the sodium salt of I about 1.0 percent by weight of the sodiumsalt of sulfated 3,668,075 I 3 4 p sulfated polyglucuronic acid methyl ester andthe sodium salt penicillin and thallium'acetate may be added to the medium. o vl lllfated polymannuronic acid methyl glycoside. The spe- Their addition is optional and not necessary to the practice of cies M. pulmonis; M. orale. ype 2 and M. fermentaru' are not this invention. Generally, penicillin is added in sufiicient able to growin the presence of the former-while the species M. quantities to provide from about 100 to about 1,000 units per pulmoms and M. omle, type 2 are not able to grow in the 5 ml. of Moreover, about 1 part by weight of thallium presence of'the latter compound. 'By using both the disc acetate per 2,000 parts by volume of medium is ordinarily technique and .the medium method, one can facilitate the used. This medium supports the growth ofall recognized identification of a particular mycoplasma isolate according 'to human large colony mycoplasma strains.

. glycosi .+=iuhlbition (no growth).

the following table; As indicated heretofore, the preaent'invention provides a INHIBITION OF:

h I I r M. M. g M. M;

' M. hominis 'ALhominir, M. orale, M. orale, fersulipncupul- Compound (sodium salt of) v Method type 1 type 2 type 1 type 2 mentam varium monlae mantis Polyauetholsulfonate Disc 0 suliated-polymannuronic acid .do 0 0 0 0 Sulfated lyglucuronic acid methyl Medium-.. 0 0 0 0 0 0 I 0 o 0 1 o 0 er. Sulfate dpolymannuronic acid methyl do 0 e. v

i The manner in which the present invention is carried out significant diagnostic tool. By using the aforementioned will bereadily appreciated by persons skilled in the art. In the heparinoids in the manner described herein, one can facilitate disc technique, the heparinoid compound is first dissolved in the identification of human'mycoplasma species. Certain spewater orin saline solution. The concentration of the solution, cies of mycoplasmas can be identified specifically, e.g., M. .thus prepared,is not particularly critical. However, there is no hominis, type 1 and M. salivarium and M. fermions. The

particular advantage in using aso l ution containing greater other species are differentiated into, groups for further than about 5.0 percent by weight of the sodium salt of delineation by other diagnostic tcsls..Furthermor'e, utilizing polya'nethol sulfonate or a solution containing more than the heparinoid compounds which inhibit the growth thereof,

polymannuronic acid. In generaL-a solution c ntai ing from can be eliminated from tissue cultures. This can be accomabout 0.5 percent to about 5.0 percent by weight of .the lished by adding the heparinoid compound or compounds to hep rin mp will be mp y The 'wllllion. thus the tissue culture at a final concentration of 0.05 to obtained, is used to moisten paper discs having a diameter, for 1,0 r e t b wei ht,

. mg. of heparinoid compound per disc. The discs are placed on are not t be a senge,

example, of from about 6 mm. to about .8 mm. Preferably, For a fuller understanding of the nature and objects of this each disc is moistened with from about 0.01 ml. to about 0.03 ml. of the solution to provide from about 0.1 mg. to about 1.0 whi h re given merely as an illustration of the invention and the agar plates which contain a conventional EXAMPLE 1 venient form of the reagent.

which is inoculated with the mycoplasma isolate. The discs 40 h can be placed on the plates while still moist or, in the alternaa p e. f y I l tive, the moistened discs may be dried before use. in the dry layer conventional e Pl r s a diameter 0 state they have the advantage of providing astable and conm- The miidium emulated y P f g a I hour broth culture ofM. type i thereon. After allow-' in those cases where the disc method is not employed, i.e., S inoeulum to 'yr p p efleh hlvinl d lmehere th heparinbid o ds in use are he s di l r ter of approximately 6.5mm. were placed onthe plate in coni ulf t dl d m i acid. methyl ester and/0|- the sodium tact with the inoculated medium. One of the discs had been several mycoplasma especiallyM. hominis, type 1,

invention, reference may be had to the following examples salt of' sulfated polymannuronic acid methyl glycoside,-the previelely saturated with the sodium-salt of polyaneflrbl -r heparinoid is added to the culture mediumihgredients. This is fonale, While the r ad een Saturated With-the sodium' true also in the case ofthe sodium saltof polyanethol sul- 5o salt of sulfated polymannuronic acid. The respective salts fonate the sodium salt of sulfated polyma'nnuronic acid were introducedonto the in the form of 1.0 percent-by whichare inhibitors to the aforementioned mycoplasrnas by weight solutions in saline. A sufficient quantity (0.02 ml.) of

i either the disc or direct addition to the medium technique. solution was usedin each instance to provide the discs with The quantity of heparinoid compound which is incorporated approximately 0.2 mg. of the heparino'id compound.

into the'mediurn is not critical. Generally, however, from The medium used in the example, namely, Hayfliclt mediabout 0.5 percentto about 1.0 percent by weight of the um, was composed of a basic PPLO medium, i.e., 700 ml. of

i .heparinoid compound will be incorporated into the medium. Difco Bacto-PPLO Agar, to which was added 100 ml. of The he'parinoid may be incorporated into either a broth pooled horse serum and 100 ml. of 25 percent extract of fresh (liquid) medium or an agar (gel) medium. Generally, it is bakers yeast. To thismedium there was added 200 units per preferred to incorporate the heparinoid in an agar medium, ml. of penicillin and 1 part thallium acetate per 2,000 units of I which is distributed in plates, allowed to solidify and, medium. The latter ingredients were to avoid bacterial thereafter, inoculated with the mycoplasma isolate and incucontamination.

I A control plate is prepared in amanner identical tothe The plate was allowed to incubate at'37 C. in a humid I test plate except that the heparinoid compound is not incore 5 chamber containing an atmosphere of air. Periodically they porated intothe medium. 6 were observed under microscope at to 100 times magln general, any medium in which mycoplasmas are conven- I nification for the formation of mycoplasma colonies. it was tionally grownis used in the practice of the present invention. found that a zone of inhibition formed around the disc satuin the preferred embodiment of the in ntio however, the rated with the sodium salt of polyanethol sulfonate and that a V a Mum of Hayflick "Win15! iHSMInPIOSled- Thiezone of inhibition also formed aroundthe disc saturated with utilizes a bsic PPLO medium, which is prepa ed, the sodium salt of sulfated polymannuronic acid. The forma- I I e.g.,- by dissolving 23.8 grams of Difco Bacto-Pl-"LO Agar tion of the zone of inhibition around each disc indicates that dehydrated in 700 ml. water. This medium is sterilthe sodium salt ofpolyanethol sulfonate andthe sodium salt of ind by autoclaving at 15 pounds pressure for 15 minutes and sulfated polymannuronic acid each inhibit the growthof the is supplemented with 200 ml. of pooled horse serum and 100 mycoplasrna M. hominir, type 1.

ml. of a 25 percent aqueous extract of fresh baker's yeast. A series of experiments were carried out, in the manner .Furthennore, in order to prevent bacterial contamination, described heretofore using, in' lieu of the M.'hominis, type i.

containing broth, broths containing the mycoplasmas M. homim's, type 2 (M. arthriridils); M. orale, type 1; M. arale, type 2; M. fermentans; M. salivarium, M. pneumoniae; and M. pulmonir. The plates were incubated under appropriate conditions and for a sufficient length of time to obtain optimal growth. It was determined that both of the aforementioned heparinoid compounds inhibited the growth of the mycoplasmas M. orale, type 2; M. pulmom's and M. fermentans. The growth of the mycoplasmas M. hominis, type 2; M. pneumoniae and M. orale, type 1 was only inhibited by the disc containing the sodium salt of polyanethol sulfonate. M. salivan'um was the only species not inhibited by either hepariple l to be active as mycoplasma growth inhibitors by the disc method, were also tested to determine if they inhibited mycoplasma growth when added directly to the medium. It was found that, in the case of each compound, the mycoplasma, whose growth was inhibited when tested by the disc method, was unable to grow in the inhibitor-containing medium.

The following table summarizes the results of these experinoid. ments:

INHIBITION OF:

M. hominls, M. lloml'nis, M. orale, M. orale, M. fer- M. snli- .ll. pneu- .ll. pul- Compound (sodium salt of) Method type 1 type 2 typo 1 type 2 mentans t'arium maniac monis Polyanetholsullonate Mediu1n sulfated polymannuronic acid do 0 0 0 0 Sullpted polyglucuronic acid methyl do 0 0 0 0 0 es er. sulfated polymannuronic acid methyl -..do 0 0 0 0 0 0 glycoside.

+=lnhibition (no growth). O=N0inl1ibition (growth). kw NH H I v In the table which follows hereinafter, there is set forth, in The sodium salt of polyanethol sulfonate used herein was mm., the zone of inhibition which formed when 6.5 mm. discs saturated with the aforementioned heparinoid compounds were placed in contact with plates of l-layflick agar medium inoculated with appropriate mycoplasma-containing broths:

ZONE OF INHIBITION produced as described in, Example 1 of us. Pat. NO.

1,907,371. The sodium salt of sulfated polymannuronic acid methyl glycoside used herein was produced as described in Example 5 of us. Pat. No. 2,694,058.

-J M. homim's. M. hominis, M. fer- M. orale, M. orale, M. sali- M. pul- M. pneu- Compound type 1 type 2 menhms type 1 type 2 varium mom's .moniae 12 11 14 12 15 0 21 8 0 l5 0 0 13 0 Zone size includes diameter of disc (6.5 mm.). A=Sodium salt of polyanetholsulfonate (5% on disc).

amasqtsusa qsosmw sisvw q 0 on disc):

EXAMPLE 2 For the sake of completeness, the preparation of the sodium salt of sulfated polyglucuronic acid methyl ester and the In this example, ml. of Hayflick agar medium containing 0.5 percent by weight of sodium salt of sulfated polyglucuronic acid methyl ester was first prepared. In this preparation, 0.25 gram of sodium salt of sulfated polyglucuronic acid methyl ester and 1.2 gram of Bacto-PPLO Agar was added to 35 ml. of distilled water. The mixture was heated to dissolve the ingredients and the solution, thus formed, was sterilized by autoclaving at 15 pounds pressure for a period of about 15 minutes. At the end of that period of time, the medium was allowed to cool to a temperature of about 45 C.,,following which 10 ml. of sterile horse serum and 5 ml. of fresh, sterile 25 percent yeast extract was aseptically added. Penicillin G was then addedto the mixture to a final concentration of 200 units per ml.

While being maintained at a temperature of 43 to 45 C., the medium, prepared as described in the preceding paragraph, was poured into a 100 mm. standard plastic Petri dish and the medium was allowed to solidify at room temperature. The plate was then divided into eight sectors. The medium in each sector was then inoculated with 0.01 ml. of a 48 hour to 96 hour broth culture (5 day culture in the case of M. pneumoniae) of a different mycoplasma species and incubated for a suitable period at 37 C. The ability of the sodium salt of sulfated polyglucuronic acid methyl ester to inhibit the growth of the mycoplasma species M. hominis, type 1, M. hominid", type 2, M. pulmonis, M. fermentans, M. pneumoniae, M. salivan'um, M. orale, type I and M. orale, type 2 was determined. It was found that, of the mycoplasma species tested, the only species which were not able to grow in the presence of the sodium salt of sulfated polyglucuronic acid methyl ester were the species M. pulmonis, M. fermentans and M. orale, type 2.

The foregoing procedure was repeated using the sodium salt of sulfated polymannuronic acid methyl glycoside as the growth inhibitor. It was determined that the only mycoplasma species which was not able to grow in the presence of the sodipreparation of the sodium salt of sulfated polymannuronic acid is set forth hereinafter. It should be understood, however,

PREPARATION OF THESODIUM SALT OF SULFATED POLYMANNURONIC ACID a. Preparation of Alginic Acid Fifty grams of commercial sodium alginate was suspended in one liter of warm water in a Waring blender and stirred to form a clear thick gel. Thereafter, 1.0 N hydrochloric acid was added to the gel with stirring, until acid to Congo red pH about 3.5). The precipitate was allowed to settle and the supernatant liquor was siphoned off. The precipitate was washed with three 500 cc. portions of ethanol while in the blenderwith stirring and siphoning as previously. Finally, the amorphous powder is collected on a sintered glass funnel, washed with ethanol, and dried in high vacuo over anhydrous calcium chloride. By the described procedure, alginic acid was obtained as a dry free-flowing powder. b. Sulfation of Alginic Acid A mixture of chlorosulfonic acid and pyridine was prepared at a temperature of 0 to 5 C. by the careful addition of 140 cc. of chlorosulfonic acid to 700 cc. of dry pyridine. 20 Grams of powdered alginic acid was then added while keeping the temperature between 0 and 10 C. After 'the addition, the mixture was stirred for onehour at room temperature, following which it was heated with constant stirring at 70 to C. for 6 hours. The reaction mixture was then'allowed to stand at room temperature ovemight. The crude pyridine salt of the sulfated alginic acid was isolated by heating the reaction mix- The pyridine salt was purified by dissolving it in 150 cc. of water and the solution was clarified by filtration through an asbestos pad. The purified pyridine salt was isolated by the addition of the filtrate to 1,500 cc. of methanol with constant stirring.

The purified pyridine salt, produced as described in the g. preceding paragraph, was converted to the sodium salt by dissolving it in 100 cc. of water, cooling the solution to 5 C. and adding 6 N sodium hydroxide, while maintaining the temperature at about 5?. C., until the pH of the solution was about 9.5 The alkaline solution was then added rapidly to 10 volumes of methanol with constant stirring to precipitate the sodium salt. The salt obtained was purified further by dissolving it in water and precipitating it with methanol as previously described. The sodium salt of the sulfuric acid ester of polymannuronic acid (alginic acid) was obtained as a light yellow free-flowing powder when dried in high vacuo over anhydrous CaCl,.

PREPARATION OF SODIUM SALT OF SULFATED POLYGLUCURONIC ACID METHYL ESTER a. 50 Grams of commercial oxidized cellulose (IO-l2 percent carboxyl) is suspended in 500cc. of 5 percent l-lCl in methanol and the mixture stirred under refiux for 96 hours. The mixture was then cooled and the precipitate was collected by centrifugation and washed with several volumes of methanol and finally dried over anhydrous calcium chloride in vacuo. There was obtained a degraded oxidized cellulose ester. 1

b. To a mixture of 74 cc. of chlorosulfonic acid and 430 cc. of dry pyridine prepared at to C. was added, with constant stirring, 20 grams of the degraded oxidized cellulose ester produced as described in the preceding paragraph. The mixture was heated with stirring at 80 to 85 C. for 6 hours. The reaction mixture was allowed to stand at room temperature for 17 hours. The crude pyridine salt of the sulfated product was isolated from the reaction mixture by adding the warmed reaction mixture (60 to 70 C.) to 1.5 liters of methanol with stirring. The crude pyridine salt that separates was collected by centrifugation and washed by trituration and decantation with several volumes of methanol. To further purify the pyridine salt it was dissolved in 100 cc. of water and clarified by filtration through an asbestos pad and the purified pyridine salt recovered by precipitation with methanol as previously described.

The pyridine salt was collected by centrifugation and washed with several volumes of methanol and finally dried over calcium chloride in vacuo. The pyridine salt was converted to the sodium salt by dissolving it in 100 cc. of water and adding 6N NaOH until the pH was 9.5, the temperature being maintained between 5 and C. during the addition of the alkali. The alkaline solution was then added to 10 volumes of methanol to precipitate the sodium salt. The salt was collected by centrifugation and washed with several volumes of methanol and further purified by dissolving it in 100 cc. of water, filtering it through as asbestos pad, and reprecipitating the purified sodium salt with 10 volumes of methanol. The salt is collected, as previously described, by centrifugation, washed with methanol and finally dried in vacuo over anhydrous calcium chloride.

The product, thus obtained, was the sodium salt of sulfated polyglucuronic acid methyl ester having a viscosity of 0.185 cps.

We claim:

l. A composition for selectively inhibiting the growth of omle, type 1; and mixtures thereof,

mycoplasrnas in culture media comprising (i) a broth or agar medium and (2) a compound selected from the group consisting of a salt of polyanethol sulfonic acid, a salt of sulfated polymannuronic acid, a salt of sulfated polymannuronic acid methyl glycoside and a salt of sulfated polyglucuronic acid methyl ester, the said compound comprising from about 0.5 percent to about 1.0 percent of the weight of said composition.

2. A composition for selectively inhibiting the growth of mycoplasmas in culture media comprising a disc impregnated with a compound selected from the group consisting of a salt of polyanethol sulfonic acid and a salt of sulfated polymannuronic acid, there being present on such disc from about 0.1 mg. to about 1.0 mg of the compound.

3. A process for selectively inhibiting the growth of mycoplasmas selected from the group consisting of M. hominis, type 1; M. homim's, type 2 (M. arthrin'dir); M. fermentans; M. pulmoms; M. pneumoniae; M. orale, type 2; M.

which comprises contacting a broth or agar medium containing the mycoplasma with a salt of polyanethol sulfonic acid, there being used in said process, based on the weight of the medium, from about 0.5 percent to about 1.0 percent of the polyanethol sulfonic acid salt, incubating at about 37 C. and observing the growth of the culture.

4. A process of claim 3 wherein the salt of polyanethol sulfonic acid is introduced into the medium impregnated on a 5. The process of claim 4 wherein the disc contains from about 0.1 mg. to about 1.0 mg. of the salt of polyanethol sulfonic acid. Y

6. A process for selectively inhibiting the growth of a mycoplasma selected from the group consisting of M. hominir, type 1; M. orale, type 2; M. fermentans; M. pulmonis; and mixtures thereof which comprises contacting a broth or agar 1.0 percent of the sulfated polymannuronic acid salt, incubating at about 37 C. and observing the growth of the culture.

7. The process of claim 6 wherein the salt of sulfated polymannuronic acid is introduced into the medium impregnated on a disc.

8. The process of claim 7 wherein the disc contains from about 0.1 mg. to about 0.2 mg. of the salt of sulfated polymannuronic acid. 7

9. A process for selectively inhibiting the growth of a mycoplasma selected from the group consisting of M. fermentans; M. tacting a broth or agar medium containing the mycoplasma' with a salt of sulfated polyglucuronic acid methyl ester, thei'e being used in said process, based on the weight of the medium, from about 0.5 percent to about 1.0 percent of the sulfated polyglucuronic acid methyl ester salt, incubating at about 37 C. and observing the growth of the culture.

10. A process for selectively inhibiting the growth of the mycoplasma species M. pulmonis and M. omle, type 2 which comprises contacting a broth or agar medium containing the mycoplasma with a salt of sulfated polymannuronic acid methyl glycoside, there being used in said process, based on the weight of the medium, from about 0.5 percent to about 1.0 percent of the sulfated polymannuronic acid methyl glycoside salt, incubating at about 37 C. and observing the growth of the culture. I Y x asses omle, type 2; and M. pulmonis which comprises con- 

2. A composition for selectively inhibiting the growth of mycoplasmas in culture media comprising a disc impregnated with a cOmpound selected from the group consisting of a salt of polyanethol sulfonic acid and a salt of sulfated polymannuronic acid, there being present on such disc from about 0.1 mg. to about 1.0 mg of the compound.
 3. A process for selectively inhibiting the growth of mycoplasmas selected from the group consisting of M. hominis, type 1; M. hominis, type 2 (M. arthritidis); M. fermentans; M. pulmonis; M. pneumoniae; M. orale, type 2; M. orale, type 1; and mixtures thereof, which comprises contacting a broth or agar medium containing the mycoplasma with a salt of polyanethol sulfonic acid, there being used in said process, based on the weight of the medium, from about 0.5 percent to about 1.0 percent of the polyanethol sulfonic acid salt, incubating at about 37* C. and observing the growth of the culture.
 4. A process of claim 3 wherein the salt of polyanethol sulfonic acid is introduced into the medium impregnated on a disc.
 5. The process of claim 4 wherein the disc contains from about 0.1 mg. to about 1.0 mg. of the salt of polyanethol sulfonic acid.
 6. A process for selectively inhibiting the growth of a mycoplasma selected from the group consisting of M. hominis, type 1; M. orale, type 2; M. fermentans; M. pulmonis; and mixtures thereof which comprises contacting a broth or agar medium containing the mycoplasma with a salt of sulfated polymannuronic acid, there being used in said process, based on the weight of the medium from about 0.5 percent to about 1.0 percent of the sulfated polymannuronic acid salt, incubating at about 37* C. and observing the growth of the culture.
 7. The process of claim 6 wherein the salt of sulfated polymannuronic acid is introduced into the medium impregnated on a disc.
 8. The process of claim 7 wherein the disc contains from about 0.1 mg. to about 0.2 mg. of the salt of sulfated polymannuronic acid.
 9. A process for selectively inhibiting the growth of a mycoplasma selected from the group consisting of M. fermentans; M. orale, type 2; and M. pulmonis which comprises contacting a broth or agar medium containing the mycoplasma with a salt of sulfated polyglucuronic acid methyl ester, there being used in said process, based on the weight of the medium, from about 0.5 percent to about 1.0 percent of the sulfated polyglucuronic acid methyl ester salt, incubating at about 37* C. and observing the growth of the culture.
 10. A process for selectively inhibiting the growth of the mycoplasma species M. pulmonis and M. orale, type 2 which comprises contacting a broth or agar medium containing the mycoplasma with a salt of sulfated polymannuronic acid methyl glycoside, there being used in said process, based on the weight of the medium, from about 0.5 percent to about 1.0 percent of the sulfated polymannuronic acid methyl glycoside salt, incubating at about 37* C. and observing the growth of the culture. 